Methods of treating pseudomonas aeruginosa respiratory infections

ABSTRACT

Pseudomonas aeruginosa (PA) leads to chronic respiratory infections especially in patients with cystic fibrosis patients and chronic obstructive pulmonary disease (COPD), characterized by a high morbidity. After screening Lactobacilli coming from CF expectoration, on their capacity to inhibit two Pseudomonas aeruginosa (PA) virulence factors (elastase, pyocyanin), the inventors evaluated the effect of intranasal administration of Lactobacilli on PA murine pneumonia. The primary outcome was the bacterial lung load 24 hours after PA induced pneumonia. To understand the role of Lactobacillus, the chemokines, the pro and anti-inflammatory BAL rates were also measured. The administration of Lactobacilli cocktail 18 h prior the PA lung infection decreases significantly the lung bacterial load at 24 h post-infection. Although the mechanisms need to be deeply explored, an immunomodulation effect may be involved, notably through the recruitment of neutrophils. Thus the present relates to a method of treating a Pseudomonas aeruginosa respiratory tract infection in a patient in need thereof comprising administering to the patient&#39;s respiratory tract a therapeutically effective amount of at least one Lactobacillus strain.

FIELD OF THE INVENTION

The present invention relates to methods of treating Pseudomonasaeruginosa respiratory tract infections.

BACKGROUND OF THE INVENTION

Pseudomonas aeruginosa (PA) leads to chronic respiratory infectionsespecially in patients with cystic fibrosis patients and chronicobstructive pulmonary disease (COPD), characterized by a high morbidity(Langan K M, Current opin Infect Dise 2015, Saiman, Clin Microbiol Rev2004); this bacteria is also the main pathogen of ventilated acquiredpneumonia (VAP), associated with a high mortality (Fujitani, Chest2011). Rates of antibiotic resistance in PA are increasing (EARSS data .. . ) leading to therapeutic deadlock.

Among the antibiotherapy alternative solutions are the probiotics,defined as live microbial food components which are beneficial for humanhealth (Erickson, J Nutr 2000; Alexandre, MMI 2013). Lactobacilli, themost studied probiotic are non-pathogenic Gram-positive bacteria, theirnatural reservoir are food (milk, cheese . . . ) and human (intestine,vagina . . . ). They can exert their beneficial effect on the hostthrough different ways especially their immunomodulatory or theirantibacterial activity (Liévin-Le Moal, Servin, Clin Microbiol Rev).Valdez et al (CMI, 2005) showed that L. plantarum inhibited two PAvirulence factors controlled by quorum sensing (elastase and biofilm).Among Lactobacilli from oral human cavities and from raws milk, 8strains were screened harbouring anti-elastase and anti-biofilmproperties (Alexandre, BMC Microbiol 2014). Khailova et al (Shok, 2013),showed that oral administration of L. rhamnosus GG improved outcome7-day survival following PA-induced pneumonia; regulatory T cells mayplay a role in that protection. Randomized trials suggest thatprobiotics (one or several Lactobacillus species most of the time)decrease the incidence of VAP (Bo, Cochrane revue, 2014) but many biasare reported: single centre study, route of administration and durationof intake are different . . . (Bo, Cochrane, 2014; Cook, Trials 2016).If the oral route is often studied to analyse the Lactobacilli effect,the nasal route could provide benefits for the respiratory infection bystimulating Nasopharynx Associated Lymphoid Tissue (NALT) instead ofGALT (Kiyono, Nat Rev Immunol 2004). The intranasal administration ofLactobacillus species before intranasal inoculation of Influenza Virusor Pneumonia virus of mice decreased the mortality on these twopneumonia murine models (Izumo, Internat Immunopharmacol 2010; Dyer, Jof Virol 2016 . . . ).

SUMMARY OF THE INVENTION

The present invention relates to methods of treating Pseudomonasaeruginosa respiratory tract infections. In particular, the presentinvention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

After screening Lactobacilli coming from CF expectorations, on theircapacity to inhibit two Pseudomonas aeruginosa (PA) virulence factors(elastase, pyocyanin), the inventors evaluated the effect of intranasaladministration of Lactobacilli on PA murine pneumonia. The primaryoutcome was the bacterial lung load 24 hours after PA induced pneumonia.To understand the role of Lactobacillus, the chemokines, the pro andanti-inflammatory BAL rates were also measured. The administration ofLactobacilli cocktail 18 h prior the PA lung infection decreasessignificantly the lung bacterial load at 24 h post-infection. Althoughthe mechanisms need to be deeply explored, an immunomodulation effectmay be involved, notably through the recruitment of neutrophils.

Accordingly, a first object of the present invention relates to a methodof treating a Pseudomonas aeruginosa respiratory tract infection in apatient in need thereof comprising administering to the patient'srespiratory tract a therapeutically effective amount of at least oneLactobacillus strain.

As used herein, the term “Pseudomonas aeruginosa” or “PA” has itsgeneral meaning in the art and refers to a common Gram-negative,rod-shaped bacterium.

As used herein, the term “treatment” or “treat” refer to bothprophylactic or preventive treatment as well as curative or diseasemodifying treatment, including treatment of patient at risk ofcontracting the disease or suspected to have contracted the disease aswell as patients who are ill or have been diagnosed as suffering from adisease or medical condition, and includes suppression of clinicalrelapse. The treatment may be administered to a subject having a medicaldisorder or who ultimately may acquire the disorder, in order toprevent, cure, delay the onset of, reduce the severity of, or ameliorateone or more symptoms of a disorder or recurring disorder, or in order toprolong the survival of a subject beyond that expected in the absence ofsuch treatment. By “therapeutic regimen” is meant the pattern oftreatment of an illness, e.g., the pattern of dosing used duringtherapy. A therapeutic regimen may include an induction regimen and amaintenance regimen. The phrase “induction regimen” or “inductionperiod” refers to a therapeutic regimen (or the portion of a therapeuticregimen) that is used for the initial treatment of a disease. Thegeneral goal of an induction regimen is to provide a high level of drugto a patient during the initial period of a treatment regimen. Aninduction regimen may employ (in part or in whole) a “loading regimen”,which may include administering a greater dose of the drug than aphysician would employ during a maintenance regimen, administering adrug more frequently than a physician would administer the drug during amaintenance regimen, or both. The phrase “maintenance regimen” or“maintenance period” refers to a therapeutic regimen (or the portion ofa therapeutic regimen) that is used for the maintenance of a patientduring treatment of an illness, e.g., to keep the patient in remissionfor long periods of time (months or years). A maintenance regimen mayemploy continuous therapy (e.g., administering a drug at a regularintervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy(e.g., interrupted treatment, intermittent treatment, treatment atrelapse, or treatment upon achievement of a particular predeterminedcriteria [e.g., pain, disease manifestation, etc.]).

In some embodiments, the subject suffers from a chronic pulmonarydisease selected from the group consisting of chronic obstructivepulmonary disease (COPD), ventilated acquired pneumonia, chronicbronchitis, recurrent bronchitis, acute bronchitis, rhinosinusitis, mildpulmonary disease, hereditary emphysema, and cystic fibrosis. In someembodiments, the patient suffers from or is at risk of suffering fromcystic fibrosis. In some embodiments, the subject suffers or is at riskof suffering from a disease associated with reduced CFTR function due tomutations in the gene encoding CFTR or environmental factors (e.g.,smoke). A mutation thereof capable of regulator activity, including, butnot limited to, F508del-CFTR, R117H CFTR, and G551D CFTR (see, e.g.,http://www.genet.sickkids.on.ca/cftr, for CFTR mutations). Thesediseases include, cystic fibrosis, chronic bronchitis, recurrentbronchitis, acute bronchitis, chronic rhinosinusitis, allergicbronchopulmonary aspergillosis, bronchopulmonary aspergillosis (ABPA)and asthma. In some embodiments, the subject harbors at least onemutation in the CFTR gene, including, but not limited to F508del-CFTR,R117H CFTR, and G551D CFTR

As used herein, the term “Lactobacillus” refers to members of the genusLactobacillus, in the family Lactobacillaceae. These bacteria areGram-positive optionally anaerobic bacteria that represent a major partof the bacterial group often referred to as “lactic acid bacteria”. Thegenus includes any of the following species: Lactobacillusacetotolerans, Lactobacillus acidifarinae, Lactobacillus acidipiscis,Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus algidus,Lactobacillus alimentarius, Lactobacillus allii, Lactobacillusamylolyticus, Lactobacillus amylophilus, Lactobacillus amylotrophicus,Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus antri,Lactobacillus apinorum, Lactobacillus apis, Lactobacillus apodemi,Lactobacillus aquaticus, Lactobacillus arizonensis, Lactobacillusaviarius, Lactobacillus aviarius subsp. araffinosus, Lactobacillusaviarius subsp. aviaries, Lactobacillus backii, Lactobacillus bambusae,Lactobacillus bavaricus, Lactobacillus bifermentans, Lactobacillusbobalius, Lactobacillus bombi, Lactobacillus bombicola, Lactobacillusbrantae, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillusbulgaricus, Lactobacillus cacaonum, Lactobacillus camelliae,Lactobacillus capillatus, Lactobacillus carnis, Lactobacillus casei,Lactobacillus casei subsp. alactosus, Lactobacillus casei subsp. casei,Lactobacillus casei subsp. pseudoplantarum, Lactobacillus casei subsp.rhamnosus, Lactobacillus casei subsp. tolerans, Lactobacilluscatenaformis, Lactobacillus caviae, Lactobacillus cellobiosus,Lactobacillus cerevisiae, Lactobacillus ceti, Lactobacilluscoelohominis, Lactobacillus colini, Lactobacillus collinoides,Lactobacillus composti, Lactobacillus concavus, Lactobacillus confuses,Lactobacillus coryniformis, Lactobacillus coryniformis subsp.coryniformis, Lactobacillus coryniformis subsp. torquens, Lactobacilluscrispatus, Lactobacillus crustorum, Lactobacillus curieae, Lactobacilluscurtus, Lactobacillus curvatus, Lactobacillus curvatus subsp. curvatus,Lactobacillus curvatus subsp. melibiosus, Lactobacillus cypricasei,Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus,Lactobacillus delbrueckii subsp delbrueckii, Lactobacillus delbrueckiisubsp. indicus, Lactobacillus delbrueckii subsp. jakobsenii,Lactobacillus delbrueckii subsp. lactis, Lactobacillus delbrueckiisubsp. sunkii, Lactobacillus dextrinicus, Lactobacillus diolivorans,Lactobacillus divergens, Lactobacillus durianus, Lactobacillus equi,Lactobacillus equicursoris, Lactobacillus equigenerosi, Lactobacillusfabifermentans, Lactobacillus faecis, Lactobacillus farciminis,Lactobacillus farraginis, Lactobacillus ferintoshensis, Lactobacillusfermentum, Lactobacillus floricola, Lactobacillus florum, Lactobacillusformosensis, Lactobacillus formicalis, Lactobacillus fructivorans,Lactobacillus fructosus, Lactobacillus frumenti, Lactobacillusfuchuensis, Lactobacillus furfuricola, Lactobacillus futsaii,Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillusgastricus, Lactobacillus ghanensis, Lactobacillus gigeriorum,Lactobacillus gorillae, Lactobacillus graminis, Lactobacillushalotolerans, Lactobacillus hammesii, Lactobacillus hamsteri,Lactobacillus harbinensis, Lactobacillus hayakitensis, Lactobacillusheilongjiangensis, Lactobacillus helsingborgensis, Lactobacillushelveticus, Lactobacillus helveticus subsp. jugurti, Lactobacillusherbarum, Lactobacillus heterohiochii, Lactobacillus hilgardii,Lactobacillus hokkaidonensis, Lactobacillus hominis, Lactobacillushomohiochii, Lactobacillus hordei, Lactobacillus iners, Lactobacillusingluviei, Lactobacillus insicii, Lactobacillus intestinalis,Lactobacillus iwatensis, Lactobacillus ixorae, Lactobacillus japonicus,Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacilluskalixensis, Lactobacillus kandleri, Lactobacillus kefiranofaciens,Lactobacillus kefiranofaciens subsp. kefiranofaciens, Lactobacilluskefiranofaciens subsp. kefirgranum, Lactobacillus kefirgranum,Lactobacillus kefiri, Lactobacillus kimbladii, Lactobacillus kimchicus,Lactobacillus kimchiensis, Lactobacillus kimchii, Lactobacilluskisonensis, Lactobacillus kitasatonis, Lactobacillus koreensis,Lactobacillus kullabergensis, Lactobacillus kunkeei, Lactobacilluslactis, Lactobacillus leichmannii, Lactobacillus letivazi, Lactobacilluslindneri, Lactobacillus malefermentans, Lactobacillus mali,Lactobacillus maltaromicus, Lactobacillus manihotivorans, Lactobacillusmellifer, Lactobacillus mellis, Lactobacillus melliventris,Lactobacillus metriopterae, Lactobacillus micheneri, Lactobacillusmindensis, Lactobacillus minor, Lactobacillus minutus, Lactobacillusmixtipabuli, Lactobacillus modestisalitolerans, Lactobacillus mucosae,Lactobacillus murinus, Lactobacillus musae, Lactobacillus nagelii,Lactobacillus namurensis, Lactobacillus nantensis, Lactobacillusnasuensis, Lactobacillus nenjiangensis, Lactobacillus nodensis,Lactobacillus odoratitofui, Lactobacillus oeni, Lactobacillusoligofermentans, Lactobacillus oris, Lactobacillus oryzae, Lactobacillusotakiensis, Lactobacillus ozensis, Lactobacillus panis, Lactobacilluspanisapium, Lactobacillus pantheri, Lactobacillus parabrevis,Lactobacillus parabuchneri, Lactobacillus paracasei, Lactobacillusparacasei subsp. paracasei, Lactobacillus paracasei subsp.pseudoplantarum, Lactobacillus paracasei subsp. tolerans, Lactobacillusparacollinoides, Lactobacillus parafarraginis, Lactobacillus parakefiri,Lactobacillus paralimentarius, Lactobacillus paraplantarum,Lactobacillus pasteurii, Lactobacillus paucivorans, Lactobacilluspentosiphilus, Lactobacillus pentosus, Lactobacillus perolens,Lactobacillus piscicola, Lactobacillus plajomi, Lactobacillus plantarum,Lactobacillus plantarum subsp. argentoratensis, Lactobacillus plantarumsubsp. plantarum, Lactobacillus plantarum subsp. plantarum,Lactobacillus pontis, Lactobacillus porcinae, Lactobacillus psittaci,Lactobacillus quenuiae, Lactobacillus rapi, Lactobacillus rennini,Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rimae,Lactobacillus rodentium, Lactobacillus rogosae, Lactobacillus rossiae,Lactobacillus ruminis, Lactobacillus saerimneri, Lactobacillus sakei,Lactobacillus sakei subsp. carnosus, Lactobacillus sakei subsp. sakei,Lactobacillus salivarius, Lactobacillus salivarius subsp. salicinius,Lactobacillus salivarius subsp. salivarius, Lactobacillussanfranciscensis, Lactobacillus saniviri, Lactobacillus satsumensis,Lactobacillus secaliphilus, Lactobacillus selangorensis, Lactobacillussenioris, Lactobacillus senmaizukei, Lactobacillus sharpeae,Lactobacillus shenzhenensis, Lactobacillus sicerae, Lactobacillussilagei, Lactobacillus silagincola, Lactobacillus siliginis,Lactobacillus similis, Lactobacillus sobrius, Lactobacillussonghuajiangensis, Lactobacillus spicheri, Lactobacillus sucicola,Lactobacillus suebicus, Lactobacillus sunkii, Lactobacillus suntoryeus,Lactobacillus taiwanensis, Lactobacillus thailandensis, Lactobacillusthermophilus, Lactobacillus thermotolerans, Lactobacillus timberlakei,Lactobacillus trichodes, Lactobacillus tucceti, Lactobacillus uli,Lactobacillus ultunensis, Lactobacillus uvarum, Lactobacillusvaccinostercus, Lactobacillus vaginalis, Lactobacillus versmoldensis,Lactobacillus vespulae, Lactobacillus vini, Lactobacillus viridescens,Lactobacillus vitulinus, Lactobacillus vermiforme, Lactobacilluswasatchensis, Lactobacillus xiangfangensis, Lactobacillus xylosus,Lactobacillus yamanashiensis, Lactobacillus yamanashiensis subsp. mali,Lactobacillus yamanashiensis subsp. yamanashiensis, Lactobacillusyonginensis, Lactobacillus zeae, Lactobacillus zymae.

In some embodiments, Lactobacillus salivarius (Ls) is administered tothe patient.

In some embodiments, Lactobacillus brevis (Lb) is administered to thepatient.

In some embodiments, at least 2, 3, 4 or 5 Lactobacillus strains areadministered to the patient.

In some embodiments, Lactobacillus paracasei, Lactobacillus salivariusand Lactobacillus brevis (Lpsb or WL) are administered to the patient.

In some embodiments, Lactobacillus salivarius and Lactobacillus brevisare administered to the patient.

In some embodiments, the Lactobacillus strain is a probiotic strain. Asused herein the term “probiotic” is meant to designate livemicroorganisms which, they are integrated in a sufficient amount, exerta positive effect on health, comfort and wellness beyond traditionalnutritional effects. Probiotic microorganisms have been defined as “Livemicroorganisms which when administered in adequate amounts confer ahealth benefit on the host” (FAO/WHO 2001). As used herein theexpression “probiotic Lactobacillus strain” denotes a Lactobacillusstrain that has a beneficial effect on the health and well-being of thehost.

In some embodiments, the probiotic Lactobacillus strain of the presentinvention is a viable probiotic Lactobacillus strain. The expression“viable probiotic Lactobacillus strain” means a microorganism which ismetabolically active and that is able to colonize the respiratory tractof the subject.

Typically, the probiotic Lactobacillus strain of the present inventionis produced with any appropriate culture medium well known in the art.Various fermentation media are suitable according to the invention, suchas (but not limited to) e.g. firstly an industrial medium, in which thestrain(s) is/are grown, and that is used as is or after concentration(e.g. drying) or after addition to another food base or product.Alternatively, bacterial cells, or bacterial cells with medium (e.g. thefermentation broth), or fractions of such cell comprising medium (i.e.medium with said bacterial strain/s) may be used. The cells or the cellcomprising medium comprise live or viable bacterial cells and/or dead ornon-viable bacterial cells of the strain(s). The medium may thus betreated by, but not limited to, heating or sonication. Also lyophilized,or frozen, bacteria and/or cell-free media (which may be concentrated)are encompassed in the methods for preparing the probiotic Lactobacillusstrain of the present invention.

As used herein, the term “effective amount” refers to a quantitysufficient of the Lactobacillus strain to achieve the beneficial effect.In the context of the present invention, the amount of the Lactobacillusstrain administered to the subject will depend on the characteristics ofthe individual, such as general health, age, sex, body weight . . . .The skilled artisan will be able to determine appropriate dosagesdepending on these and other factors. For example, the Lactobacillusstrain shall be able to generate a colony is sufficient to generate abeneficial effect on the subject.

The composition comprising the effective amount of Lactobacillus mayconveniently be administered by any method that allows administration tothe respiratory tract (e.g. lungs). For example, nasal drops can beinstilled in the nasal cavity by tilting the head back sufficiently andapply the drops into the nares. The drops may also be inhaled throughthe nose. Alternatively, a liquid preparation may be placed into anappropriate device so that it may be aerosolized for inhalation throughthe nasal or buccal cavity. For administration by inhalation thecompositions may be delivered in the form of an aerosol spraypresentation from pressurized packs or a nebulizer, with the use of asuitable propellant. Administered spray and drops can be a single doseor multiple doses. These procedures may involve mixing, granulating andcompressing or dissolving the ingredients as appropriate to the desiredpreparation. It will be appreciated that the form and character of thepharmaceutically acceptable diluent is dictated by the amount ofLactobacillus active ingredient with which it is to be combined, theroute of administration and other well-known variables. The carrier(s)must be “acceptable” in the sense of being compatible with the otheringredients of the formulation and not deleterious to the recipientthereof. “Carriers” or “vehicles” mean materials suitable foradministration and include any such material known in the art such as,for example, any liquid, gel, solvent, liquid diluent, solubilizer, orthe like, which is non-toxic and which does not interact with anycomponents of the composition in a deleterious manner. Examples ofnutritionally acceptable carriers include, for example, water, saltsolutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils,polyethylene glycols, propylene glycol, liposomes, sugars, gelatin,lactose, amylose, magnesium stearate, talc, surfactants, silicic acid,viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, and the like. Spray compositions for topicaldelivery to the lung by inhalation may for example be formulated asaqueous solutions or suspensions or as aerosols delivered frompressurized packs, such as a metered dose inhaler, with the use of asuitable liquefied propellant. Aerosol compositions suitable forinhalation can be either a suspension or a solution and generallycontain the compositions of the present invention and a suitablepropellant such as a fluorocarbon or hydrogen-containingchlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes,e.g. dichlorodifluoromethane, trichlorofluoromethane,dichlorotetra-fluoroethane, especially 1, 1, 1,2-tetrafluoroethane, 1,1, 1,2,3,3,3-heptafluoro-n-propane or a mixture thereof. Carbon dioxideor other suitable gas may also be used as propellant. The aerosolcomposition may be excipient free or may optionally contain additionalformulation excipients well known in the art such as surfactants, e.g.,oleic acid or lecithin and cosolvents, e.g. ethanol. Pressurizedformulations will generally be retained in a canister (e.g. an aluminumcanister) closed with a valve (e.g. a metering valve) and fitted into anactuator provided with a mouthpiece.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1: Pulmonary PA burden measured on total lung homogenates. ControlPAO1, n=5 mice; SL (2 L. fermentum strains and 1 L. rhamnosusstrains)+PAO1, n=5 mice; WL (1 L. paracasei, 1 L. salivarius and 1 L.brevis strains)+PAO1,n=4 mice.

FIG. 2: A) and B) Total white blood cell count in BALs at 6 h and 24 hpost infection with PA. C) and D) Neutrophils ratio in BALs at 6 h and24 h post infection with PA. Statistical significance: *, p<0.05. BAL:Bronchoalveolar lavage; PA: P. aeruginosa; WBC: White blood cells.Control PAO1, n=5 mice; SL (2 L. fermentum strains and 1 L. rhamnosusstrains)+PAO1, n=5 mice; WL (1 L. paracasei, 1 L. salivarius and 1 L.brevis strains)+PAO1,n=4 mice.

FIG. 3: Survival rate of mice. Priming of the respiratory tract withL.psb (1×10{circumflex over ( )}6 CFU/mouse) resulted in survival inresponse to PA infection (2×10{circumflex over ( )}6 CFU/mouse).Statistical significance: *, p<0.001 for the L.psb+PAO1 groups comparedto the Control PAO1 group.

FIG. 4: PAO1 lung load in mice pre-treated with live Lactobacillus 24 hpost-infection. Control PAO1, n=12 mice; Lpsb (L. paracasei, L.salivarius and L. brevis) cocktail+PAO1, n=12 mice; L.paracasei+PAO1,n=7 mice; L. salivarius+PAO1,n=12 mice; L.brevis+PAO1,n=11 mice.

EXAMPLE 1

Methods

Ethics

This study is approved by our local ethics committee and the ethicscommittee for animal experiments (DAP 2017040717237994).

Bacterial Strains

Pseudomonas aeruginosa Strain:

P. aeruginosa PAO1 was chosen as reference strain for all theexperiments [1]. It was frozen at −80° C. before subculture on MuellerHilton agar plate before the experiments.

Lactobacillus Strains:

One hundred and thirty-seven Lactobacillus isolates were previouslyisolated from CF patient's respiratory samples (Fangous et al, Researchin Microbiology). Fifty Lactobacillus isolates were selected from PAcolonised (n=30) or not colonised patients (n=20), by respecting thespecies prevalence observed within each group of patients. Theseisolates were screened in vitro for their ability to decrease thesynthesis of 2 PA virulence factors, the pyocyanin and the elastase.

All isolates were frozen at −80° C. before subculture on 5% sheep-bloodagar (bioMérieux, Marcy l'Etoile, France) in 5% CO₂ at 37° C. for 2 daysbefore the experiments.

Inhibition Tests of Lactobacillus Strains on PAO1 Virulence Factors

Elastase

For the elastase assay, PAO1 and Lactobacillus isolates were cultivatedovernight separately at 37° C. in Brain Heart Infusion broth (BHI). Theinhibition of the elastolytic activity of Pseudomonas aeruginosa PAO1 byLactobacilus isolates was investigated by colorimetric assay, usingElastin Congo Red (Sigma), as adapted by Alexandre and collaborators[2]. Succinctly, overnight culture of PAO1 in BHI broth was washed twicewith isotonic saline solution and adjusted to 5×10⁷ CFU/ml in brothmedia. Overnight culture of Lactobacillus in BHI broth was neutralisedwith NaOH 0.1M and adjusted to 5×10⁷ CFU/ml in broth media. A vol/volco-culture was made and incubated 20 hours under aerobic conditions at37° C. After centrifugation (20′ at 3500 g), 50 μL of the supernatantwas mixed with 1 ml of Elastin Congo Red solution (20 mg/ml in a 10 mMsodium phosphate buffer) and incubated for 20 hours more underagitation. Finally, the soluble fraction released in the supernatant byelastase was measured at 495 nm after centrifugation (20′ at 3500 g)with a spectrophotometer.

The results were normalized to the OD₅₉₅ of the co-culture and expressedas a ratio of the absorbance observed in presence of the Lactobacillusisolate to the absorbance observed with a monoculture of PAO1.

The experiments were conducted twice to three times for each isolate ofLactobacillus.

Pyocyanin

For pyocyanin production, P. aeruginosa PAO1 was grown overnight inBacto-Peptone (BP) broth (20 mg/L BP, MgCl₂ 1.4 g/L, K₂SO₄ 10 g/L).Lactobacillus was grown overnight on MRS broth. A vol/vol co-culture wasmade as previously described for the elastase experiments, and incubateunder aerobic conditions at 37° C. The inhibition of the pyocyaninsynthesis was investigate by colorimetric assay after extraction in anacid solution as previously described [3].

The results were normalized to the OD₅₉₅ of the co-culture and expressedas a ratio of the absorbance observed in presence of the Lactobacillusisolate to the absorbance observed with a monoculture of PAO1.

The experiments were conducted twice to three times for each isolate ofLactobacillus.

Murine Model of Pneumonia:

Preparation of the Bacterial Strains

Lactobacillus were grown overnight on MRS broth under aerobic conditionsat 37° C. The 3 strains of Lactobacillus with the better inhibitiveabilities against P. aeruginosa PAO1 were equally mixed in a cocktailnamed “Strong Lactobacilli; SL”.

The 3 strains with the weakest abilities were mixed and named “WeakLactobacilli; WL”.

P. aeruginosa PAO1 was grown overnight on Luria Bertani (LB) broth underaerobic conditions at 37° C.

Each culture was washed twice with isotonic saline solution and adjustto 10⁸ CFU/ml for the P. aeruginosa PAO1 suspension, or to 10⁶ CFU/mlfor the SL and WL suspensions, based to the OD_(595 nm) and controlledby serial dilution and plating on Mueller Hinton agar plates intriplicates.

Animals

C57BL/6 mice, aged 6-8 weeks old, were purchased from Janvier Labs (LeGenest Saint Isle, France) and maintained at the University of Brest,France. Mice received water and food ad libitum, and were monitoredevery eight hours until being sacrificed.

Seventy-one mice were divided in 5 groups: Control, n=10; Control SL,n=16; Control PAO1, n=16; SL+PAO1, n=16; WL+PAO1, n=13.

Infection Model of Acute Pneumonia

Bacteria were administrated by intranasal instillation of 20 μL of thebacterial suspension (10 μL per nostril), under a short intraperitonealanaesthesia with ketamine/xylazine (100/10 mg/kg) allowing maintenanceof spontaneous breathing.

Lactobacillus suspension (SL or WL) was administrated 18 hours prior theinfection with P. aeruginosa PAO1.

Control groups received 20 μL of isotonic saline suspension instead ofLactobacillus suspension and/or P. aeruginosa suspension.

Sampling Procedure

Six or 24 hours post infection with PAO1, mice were anesthetized withintraperitoneal injection of ketamine/xylazine (100/10 mg/kg) andeuthanasied by intracardiac exsanguination.

Blood, bronchoalveolar lavage (BAL), lung and spleen tissues wereharvested from animals under aseptic conditions.

BAL was performed after euthanasia by cannulation of the trachea andinjection and aspiration of 500 μl of isotonic saline solution threetimes.

Bacterial Loads

The lungs were removed and homogenized with 4 ml of isotonic salinesolution with Ultra-Turrax. Bacterial loads of PAO1 and Lactobacilluscocktails were determined by plating serial dilutions of total lunghomogenate on Cetrimide and MRS agar plate. Each dilution was plated induplicate. Plates were incubated 24 to 48 h at 37° C. under aerobicconditions. Colonies were confirmed using MALDI-TOF mass spectrometry(Microflex LT, Bruker Daltonics, Bremen, Germany). Identifications wereobtained when scores were strictly superior to 2.

White Blood Cells (WBC) Count

The total white blood cells count was enumerated by manual countingmethod with a hemocytometer by light microscopy.

Macrophages, neutrophils and lymphocytes were differentiated aftercentrifugation, cytospins preparation and May-Grünwald-Giemsa staining.

Cytokine Measurement

The concentrations of the cytokines were performed on BAL supernatant,after centrifugation at 4° C., and freezing at −80° C.

The cytokines studied were IL-1b, IL-12, IL-17-a, IL-22, IL-23, IFN-gand the 2 chimiokines CXCL-1 ad CXCL-2. Dosages were performed withsingle Elisa kits.

Statistics

Results are presented as mean and standard error of the mean.Comparisons between the groups were analysed by the Mann-Whitney test.Results were considered statistically significant for p<0.05. Allstatistical tests were performed using the R software.

Results

Screening In Vitro of Lactobacillus Strains

The 50 selected strains were distributed as figured on the Table 1.Eleven species were represented. Considering the anti-elastolyticactivity, 25 (83%) strains from PA colonized patients and 15 (75%)strains from PA non colonized patients exhibited an anti-PA activitywith respectively average of 60.6±0.15% and 64.7±0.15% of activity.

Considering the inhibition of the pyocyanin synthesis, 6 (20%) strainsfrom PA colonized patients and 6 (30%) strains from PA non colonizedpatients exhibited an anti-PA activity with respectively average of83.6±0.15% and 80.15±0.17% of activity.

To constitute 2 blends of Lactobacillus to administrated to our micemodel of PA pneumonia, 3 strains with the better anti-PA activities and3 strains with the weakest anti-PA activities were selected.

The “Strong Lactobacilli” (ST) blend was constituted with 2 L. fermentumstrains and 1 L. rhamnosus strains. The three strains were from PAcolonized patients.

The “Weak Lactobacilli” (WK) blend was constituted with 1 L. paracasei,1 L. salivarius and 1 L. brevis strains. The last two strains were fromPA colonized patients.

The anti-PA activity of these 6 strains are shown on the Table 2.

Administration of Lactobacilli Decreases the Lung PA Load

Significant decreases of 2 and 4 log of PAO1 were observed 24 h afterPAO1 instillation in SL+PAO1 (4.6.10¹ CFU/g) and WL+PA01 (<1 CFU/g)groups compared to PAO1 group (6.8.10⁴ CFU/g) (FIG. 1). No increase inLactobacilli load was observed whatever the group studied. However,Lactobacilli were still present in the lung 24 h after the instillation,with 1.16.10⁴ and 1.13.10³ CFU/g for the SL+PA01 and WL+PA01 groupsrespectively.

BAL Cytological Analysis

An increase of the WBC count and PNN were observed following theadministration of the SL cocktail in the Control SL group, butsignificantly less important than in the Control PAO1 group (FIG. 2). Asignificant decrease of PNN in BAL was observed 6 h and 24 hpost-infection in the two groups receiving prophylactic administrationof Lactobacillus (SL+PAO1 and WL+PA01 groups) compared to PAO1 group(FIG. 2).

BAL Cytokine Analysis

Following the decrease of the recruitment of PNN due to prophylacticadministration of Lactobacilli cocktail, the immunological response wasstudied through cytokines and chemokines dosages 6 h (T6) and 24 h (T24)post PAO1 administration.

No production of CXCL1 and CXCL2 was observed in the Control and ControlSL at T6 and T26 whereas an important increase was observed at Thin theControl PAO1 group. The chemokines CXCL1 and CXCL2 BAL's levels weredecreased in SL+PAO1 (DNS) and WL+PA01 groups (DS) at 6 h post-infectioncompared to the Control PAO1 group.

No difference of production of IL-1B was observed between the Controland Control SL groups at T6 and T24. However, an increase was observedat T6 in the Control PAO1 group (mean=115 pg/ml). The IL-1B BAL's levelwere decreased in SL+PAO1 (mean=67.5 pg/ml) and WL+PA01 groups(mean=33.6 pg/ml; p=0.01) at 6 h post-infection compared to the ControlPAO1 group. A significant decrease was also observed at T24 in theSL+PAO1 (mean=9.0 pg/ml; p=0.01) compared to Control PAO1 (mean=22.2pg/ml).

No difference of production of IL-12 was observed between the Control,Control SL and Control PAO1 groups at T6. However, a slight increase wasobserved at T24 in the Control SL compared to the Control group, and asignificant increase was observed in the PAO1 group (mean=105.5 pg/ml).No clear difference was observed in the SL+PAO1 and WL+PAO1 groupscompared to Control PAO1 at T6 and T24.

No production of IFN-G was observed in the Control, Control SL andControl PAO1 at T6. However, an increase was observed in the ControlPAO1 group at T24 (mean=77.7 pg/ml) compared to the 2 others. Followingthe administration of Lactobacilli cocktail, a decrease of IFN-G wasobserved at T24 in the SL+PAO1 (mean<15 pg/ml) and WL+PAO1 (mean<15pg/ml) groups.

Finally, the immunomodulation mediated via the TH17 axis was exploredthrough IL-17A and IL-23A dosages. No production of IL-17A was observedin the Control, Control SL and Control PAO1 at T6. However, an increasewas observed in the Control PAO1 group at T24 (mean=96.4 pg/ml) comparedto the 2 others. Following the administration of Lactobacilli cocktail,a decrease of IL-17A was observed at T24 in the SL+PAO1 (mean<5 pg/ml)and WL+PAO1 (mean=3.95 pg/ml) groups. A slight production of IL-23 wasobserved whatever the group at T6 and T24, with no significantdifference following the prophylactic administration of Lactobacillicocktails.

Example 2

Methods

Ethics

This study has been approved by the ethics committee for animalexperiments (DAP2017040717237994 and DAP2017110311134961).

Preparation of the Bacterial Strains

Lactobacilli were grown overnight on MRS broth under aerobic conditionsat 37° C. Three strains without inhibitory activity were mixed as acontrol in a blend named “L.psb”. PAO1 was grown overnight inLuria-Bertani broth (Sigma) under aerobic conditions at 37° C. Eachculture was washed twice with isotonic saline solution (SS) and adjustedto 108 CFU.ml-1 for the PAO1 suspension, or to 107 CFU.ml-1 for theL.psb suspensions, based on the OD595 nm and controlled by serialdilution and plating on MH in triplicates.

Animals

C57BL/6 mice, aged 6-8 weeks old, were purchased from Janvier Labs (LeGenest Saint Isle, France) and maintained at the University of Brest,France. Mice received water and food ad libitum, and were monitoredevery eight hours until being sacrificed.

9 groups of C57BL/6 mice: Control PAO1, Control Lpsb (L. paracasei, L.salivarius and L. brevis), Control Lp (L. paracasei), Control Ls (L.salivarius), Control Lb (L. brevis), Lpsb+PAO1, Ls+PAO1 and Lb+PAO1.

Infection Model of Acute Pneumonia

Bacteria were administrated by intranasal instillation of 20 μL of thebacterial suspension (10 μL per nostril), under a short intraperitonealanaesthesia with ketamine/xylazine (100/10 mg/kg) allowing maintenanceof spontaneous breathing.

Lactobacillus suspension was administrated 18 hours prior the infectionwith P. aeruginosa PAO1.

Control groups PAO1 received isotonic saline suspension instead ofLactobacillus suspension. Control L.psb groups received isotonic salinesuspension instead of PAO1.

Survival

Mice were monitored during 7 days after infection with PAO1. Fur aspect,activity, behaviour, posture, eye lids, respiration, chest sounds, andbody weight were followed frequently during the whole experiment, andscored from 1 to 4 according to the M-CASS scoring system [17]. Whenmice reached a score of 11 during day, buprenorphine was administeredsubcutaneously (0.05 mg/kg/12 h) for analgesia. Mice were sacrificedwhen they reached a score of 4 in the 8 parameters during the day or inone parameter at night to prevent overnight death.

Sampling Procedure

Six (T6) or 24 hours (T24) post infection with PAO1, mice wereanesthetized with intraperitoneal injection of ketamine/xylazine (100/10mg/kg) and sacrificed by intracardiac exsanguination. Blood, BAL, lungand spleen tissues were harvested from animals under aseptic conditions.BAL was performed after euthanasia by cannulation of the trachea andinjection and aspiration of 500 μl of SS three times.

Bacterial Cell Count in Lung Homogenates

Mice were sacrificed at T24 and lungs removed and homogenized with SSwith Ultra-Turrax. Bacterial loads of PAO1 and Lactobacillus blends weredetermined by plating serial dilutions of total lung homogenate onCetrimide (bioMérieux) and MRS agar plates. Each dilution was plated induplicate. Plates were incubated 24 to 48 h at 37° C. under aerobicconditions. Colonies identification was confirmed using MALDITOF massspectrometry (Microflex LT, Bruker Daltonics, Bremen, Germany).

White Blood Cells Count

The total white blood cells (WBC) count on BAL was enumerated by manualcounting method with a hemocytometer (Kova slide) by light microscopy.Alveolar macrophages (AM), polymorphonuclear (PMN) and lymphocytes weredifferentiated after centrifugation, cytospins preparation andMay-Grünwald-Giemsa staining.

Cytokine Measurement on BAL

The cytokines studied were IL-1β, IL-6, IL-1β, TNF-α, and the 2chemokines CXCL-1 and CXCL-2. IL-1β, IL-6 and IL-10 (eBiosciences),TNF-α and the chemokines CXCL1 and CXCL2 (R&D System, Abingdon, UK) weredetermined in the BAL by enzyme-linked immunosorbent assay (ELISA),using commercial kits according to the manufacturer's recommendations.The lower levels of detection were 7 pg/ml for CXCL1 and CXCL2, 4 pg/mlfor IL-1β and IL-6, 8 pg/ml for IL-10 and TNF-α.

Results

In Vitro Screening of Lactobacilli Isolated from CF Respiratory Samples

Forty strains (80%) exhibited anti-elastolytic activity (meanactivity=−37.4%±0.15), and 12 (24%) exhibited anti-pyocyanin activity(mean activity=−18.13%±0.15). To constitute 2 blends of Lactobacilli toadministrate to the mice model of PA pneumonia, 3 strains with thehighest anti-PA activities (L.rff) and 3 strains with no anti-PAactivities (L.psb) were selected (Table 2).“L.rff” was constituted with 2 L. fermentum strains and 1 L. rhamnosusstrain. The three strains were isolated in PA colonised patients.“L.psb” was constituted with 1 L. paracasei, 1 L. salivarius and 1 L.brevis strains. The last two strains were isolated in PA colonisedpatients.

Nasal Priming with Lactobacilli Enhances the Survival Rate

C57BL/6 mice were inoculated intranasally with each blend ofLactobacilli 18 hours prior to PAO1 administration. All Control PAO1mice died but two (12% survival). Mice receiving L.psb were fullyprotected (100% survival) (p<0.001). None of the Control L.psb mice diednor exhibited any clinical signs of distress (FIG. 3).

Administration of Lactobacilli Decreases the Lung PA Load

Significant decrease of PAO1 lung load observed 24 h postinfection inpresence of Lpsb cocktail (9.49×102 CFU/g, p=0.010) and Ls strain(7.81×102 CFU/g p=0.011) whereas a moderate impact of Lb strain(1.16×103 CFU/g p=0.056) is observed compared to the control PAO1 group(9.34×103 CFU/g) (FIG. 4).

White Blood Cells Count and Cytokines Analysis in BAL

To elucidate the mechanism of the PA lung load reduction, weinvestigated the WBC recruitment and cytokine synthesis in the BAL (FIG.2). Control L.rff group significantly recruited more WBC at T6 and T24compared to sham mice (p<0.01). This infiltrate was mostly composed ofPMN whereas the BAL of sham mice only included AM. As expected, micefrom the Control PAO1 group exhibited a strong increased number of WBCwhich was mainly composed of neutrophils. This recruitment issignificantly more important (3 and 9 times more respectively at T6 andT24) than in the control L.rff mice. A significant decreased of PMN inBAL was observed at T6 and T24 in the L.rff+PAO1 and L.psb+PA01 groupscompared to PAO1 group. We investigated the immunological response dueto prophylactic administration of Lactobacilli blend through cytokinesand chemokines dosages in the BAL. Administration of Lactobacilli alonedid not induce the secretion of CXCL1, CXCL2, IL-1β, IL-6 and TNF-αcompared to sham mice. Infection of PAO1 induced a cytokine burstparticularly at 6 h. Prophylactic administration of Lactobacilli leadsto lower secretions of chemokines CXCL1 and CXCL2 (at T6) andproinflammatory cytokines IL-1β, IL-6 and TNF-α (both at T6 and T24) inboth L.rff+PAO1 and L.psb+PA01 groups compared to the Control PAO1 group(Data not shown). The IL-10 production was significantly increased inthe L.psb group compared to the Control PAO1 groups (Data not shown) butno difference was observed with the sham group.

DISCUSSION

Intranasal administration of Lactobacilli improves lung P. aeruginosaclearance at 24 hours post infection, associated with the decrease ofchemokines and proinflammatory cytokines, and a lower neutrophilsrecruitment.

Used as a prophylactic treatment, Lactobacilli reduce the inflammatoryresponse triggered by PA, which is deleterious on the PA control group.No difference in the PA lung load, white blood cells count or cytokinesproduction was observed between the 2 blends of Lactobacilliadministered. This might suggest that there is no correlation betweenthe anti-PA activities screened in vitro on Lactobacillus strains andtheir abilities to fight against the infection in vivo. The twovirulence factors studied for the screening, the elastase and pyocyanin,were chosen because their pathogenicity was confirmed in vivo on murinemodel of pneumonia (Le Berre et Lau, 2004). Thus, the elastolyticactivity was positively correlated to acute lung injury, and PApyocyanin deficient isogenic mutants induce less tissue damages than thewild strains. As no difference was observed in our study whatever theanti-PA activities of the Lactobacillus strains used, both virulencefactors which are quorum sensing dependent probably not interferedirectly with the innate immune response in our murine model of PApneumonia, and nor is the quorum sensing system.

Considering the different mechanisms of action of the probiotics, directantimicrobial activity, reinforcement of the epithelium barrier functionand immunomodulation (Alexandre, 2014), other hypothesis than theinhibition of the PA virulence factors must be formulated to understandhow Lactobacilli may act against PA infection. Several publicationsstudying the effect of Lactobacillus administration by oral gavagesuggest that the probiotic protective abilities of Lactobacillus arebased on the immunomodulation mediated by the gut-lung axis. However, inour study, Lactobacilli were administrated intranasally to liberate fromthis axis and observed their action on the respiratory tract whendirectly administered in situ. Nevertheless, similarities probablyexist, as the microbiota modification occurring in the gut and leadingto the immunomodulation which certainly occurs in the lung too.Lactobacilli could sufficiently stimulate the respiratory mucosal immunesystem to protect from bacterial infection.

In our study, the PA clearance lead by the Lactobacilli may be based onthe modulation of the bactericidal activity and phagocytosis activity ofthe alveolar macrophage and neutrophils, which recruitment is decreasedand associated with a lightest production of chemokines whenLactobacilli were administrated compared to the PAO1 group.

CONCLUSION

The administration of Lactobacilli cocktail 18 h prior the PA lunginfection decreases significantly the lung bacterial load at 24 hpost-infection. Although the mechanisms need to be deeply explored, animmunomodulation effect may be involved, notably through the recruitmentof PNN.

Tables:

TABLE 1 Lactobacillus strains selected for the in vitro screeningPatients status of PA colonization Species Number of strains ColonizedL. rhamnosus 9 L. fermentum 6 L. paracasei 5 L. gasseri 3 L. salivarius1 L. crispatus 1 L. johnsonii 1 L. brevis 1 L. parabuchneri 1 L. casei 1L. plantarum 1 Non colonized L. rhamnosus 5 L. fermentum 4 L. paracasei4 L. salivarius 2 L. parabuchneri 1 L. plantarum 1 L. gasseri 2 L.johnsonii 1

TABLE 2 anti-PA activities of the 6 Lactobacillus strains selected to beadministrated to the mice (expressed as a ratio of the absorbanceobserved in presence of the Lactobacillus isolate to the absorbanceobserved with a monoculture of PAO1). Anti- Anti- elastolytic pyocyaninactivity synthesis Blend of Lactobacillus Strains of Lactobacillus (%)(%) “Strong Lactobacilli” L. rhamnosus 2C 61 71 L. fermentum 9C 50 94 L.fermentum 10C 69 76 “Weak Lactobacilli” L. paracasei 9N 114 116 L.salivarius 20C 187 150 L. brevis 24C 141 174

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

1. A method of treating a Pseudomonas aeruginosa respiratory tractinfection in a patient in need thereof comprising administering to thepatient's respiratory tract a therapeutically effective amount of atleast one Lactobacillus strain.
 2. The method of claim 1 wherein thesubject suffers from a chronic pulmonary disease selected from the groupconsisting of chronic obstructive pulmonary disease (COPD), ventilatedacquired pneumonia, chronic bronchitis, recurrent bronchitis, acutebronchitis, rhinosinusitis, mild pulmonary disease, hereditaryemphysema, and cystic fibrosis.
 3. The method of claim 1 wherein theLactobacillus strain is selected from the group consisting ofLactobacillus acetotolerans, Lactobacillus acidipiscis, Lactobacillusacidophilus, Lactobacillus agilis, Lactobacillus algidus, Lactobacillusalimentarius, Lactobacillus amylolyticus, Lactobacillus amylophilus,Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillusarizonensis, Lactobacillus aviarius, Lactobacillus bifermentans,Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei,Lactobacillus coelohominis, Lactobacillus collinoides, Lactobacilluscoryniformis subsp. coryniformis, Lactobacillus coryniformis subsp.torquens, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacilluscypricasei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillusdelbrueckii subsp delbrueckii, Lactobacillus delbrueckii subsp. lactis,Lactobacillus durianus, Lactobacillus equi, Lactobacillus farciminis,Lactobacillus ferintoshensis, Lactobacillus fermentum, Lactobacillusformicalis, Lactobacillus fructivorans, Lactobacillus frumenti,Lactobacillus fuchuensis, Lactobacillus gallinarum, Lactobacillusgasseri, Lactobacillus graminis, Lactobacillus hamsteri, Lactobacillushelveticus, Lactobacillus helveticus subsp. jugurti, Lactobacillusheterohiochii, Lactobacillus hilgardii, Lactobacillus homohiochii,Lactobacillus intestinalis, Lactobacillus japonicus, Lactobacillusjensenii, Lactobacillus johnsonii, Lactobacillus kefiri, Lactobacilluskimchii, Lactobacillus kunkeei, Lactobacillus leichmannii, Lactobacillusletivazi, Lactobacillus lindneri, Lactobacillus malefermentans,Lactobacillus mali, Lactobacillus maltaromicus, Lactobacillusmanihotivorans, Lactobacillus mindensis, Lactobacillus mucosae,Lactobacillus murinus, Lactobacillus nagelii, Lactobacillus oris,Lactobacillus panis, Lactobacillus pantheri, Lactobacillus parabuchneri,Lactobacillus paracasei subsp. paracasei, Lactobacillus paracasei subsp.pseudoplantarum, Lactobacillus paracasei subsp. tolerans, Lactobacillusparakefiri, Lactobacillus paralimentarius, Lactobacillus paraplantarum,Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus plantarum,Lactobacillus pontis, Lactobacillus psittaci, Lactobacillus reuteri,Lactobacillus rhamnosus, Lactobacillus ruminis, Lactobacillus sakei,Lactobacillus salivarius, Lactobacillus salivarius subsp. salicinius,Lactobacillus salivarius subsp. salivarius, Lactobacillussanfranciscensis, Lactobacillus sharpeae, Lactobacillus suebicus,Lactobacillus thermophilus, Lactobacillus thermotolerans, Lactobacillusvaccinostercus, Lactobacillus vaginalis, Lactobacillus versmoldensis,Lactobacillus vitulinus, Lactobacillus vermiforme, Lactobacillus zeae.4. The method of claim 1 wherein at least 2, 3, 4 or 5 Lactobacillusstrains are administered to the patient.
 5. The method of claim 1wherein Lactobacillus salivarius and Lactobacillus brevis areadministered to the patient.
 6. The method of claim 1 wherein theLactobacillus strain is a probiotic strain.
 7. The method of claim 1wherein the probiotic Lactobacillus strain is a viable probioticLactobacillus strain.
 8. The method of claim 1 wherein theadministration is performed by instillation or inhalation.